The human LX-2 HSC cell line (American Type Culture Collection) was cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Wisent, Inc.) and 1% streptomycin/penicillin antibiotics at 37°C with 5% CO₂. The medium was replaced every other day. At 80–90% confluence, cells were passaged and cells in the logarithmic growth phase were used for subsequent experiments.

Recombinant full-length human AK021443 cDNA that was cloned into the pcDNA3.1 vector (pcDNA-AK021443) was designed and synthesized by Shanghai GenePharma Co., Ltd. The pcDNA3.1 empty vector was used as a negative control (Shanghai GenePharma Co., Ltd.). Cells at the density of 2×10⁶/ml were transfected with 2 µg/ml plasmids using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. At 72 h post-transfection, cells were used for subsequent experiments.

To determine cell proliferation, cells were cultured in 96-well plates and transfected with indicated vectors. At 0, 24, 48 and 72 h post-transfection, cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, 10 µl CCK-8 working solution was added to each well and incubated for 2 h at 37°C. Absorbance was measured at a wavelength of 450 nm using a microplate reader.

Flow cytometry was performed to analyze the effect of AK021443 on the cell cycle. Briefly, LX-2 cells were fixed with 70% ethanol overnight at 4°C and incubated with 0.5 mg/ml RNaseA (Thermo Fisher Scientific, Inc.) at 37°C for 30 min. Following incubation with 25 µg/ml propidium iodide (Thermo Fisher Scientific, Inc.) on ice for 1 h in the dark, the cell cycle distribution was analyzed using a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using a flow cytometry software (iSort Automated Cell Sorter A.0; Thermo Fisher Scientific, Inc).

Total RNA was extracted from LX-2 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was reverse transcribed into cDNA (37°C for 15 min and 85°C for 5 sec) using the PrimeScript RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd.). Subsequently, qPCR was performed using the TB Green Fast qPCR mix (Takara Biotechnology Co., Ltd.). The following primers were used for qPCR: AK021443 forward, 5′-CTTGAACCCAGAAGACAGG-3′ and reverse, 5′-ATGGAACATTAGAGGTAGCAC-3′; and β-actin forward, 5′-ATCGTGCGTGACATTAAGGAGAAG-3′ and reverse, 5′-AGGAAGGAAGGCTGGAAGAGTG-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 2 min; followed by 40 cycles at 95°C for 20 sec, 58°C for 20 sec and 72°C for 20 sec. mRNA expression levels were quantified using the 2^−ΔΔCq method (18) and normalized to the internal reference gene β-actin.

Total protein was extracted from LX-2 cells using RIPA buffer (Beyotime Institute of Biotechnology) and quantified using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein (50 µg) were separated via 8–12% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with TBS-0.05% Tween-20 containing 5% skimmed milk at room temperature for 2 h. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies targeted against: cyclin D1 (cat. no. ab16663; 1:200; Abcam), cyclin-dependent kinase 2 (CDK2; cat. no. ab32147; 1:1,000; Abcam), p21 (cat. no. ab109520; 1:5,000; Abcam), E-cadherin (cat. no. ab40772; 1:10,000; Abcam), N-cadherin (cat. no. ab76011; 1:10,000; Abcam), vimentin (cat. no. ab92547; 1:5,000; Abcam), snail (cat. no. ab216347; 1:1,000; Abcam), TIMP1 (cat. no. ab211926; 1:1,000; Abcam), collagen1 (cat. no. ab34710; 1:10,000; Abcam), endothelin 1 (ET-1; cat. no. ab113697; 1:250; Abcam), matrix metallopeptidase (MMP)2 (cat. no. ab92536; 1:5,000; Abcam), MMP9 (cat. no. ab38898; 1:1,000; Abcam) and GAPDH (cat. no. sc-32233; 1:10,000; Santa Cruz Biotechnology, Inc.). Following primary incubation, the membranes were incubated with a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; 1:10,000; Abcam) at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence kit (Cytiva) and ImageJ software (v1.4; National Institutes of Health) was utilized to analyze the intensity of each protein band. GAPDH was used as the loading control.

LX-2 cells cultured on slides were fixed with 4% paraformaldehyde for 20 min at room temperature (RT), blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at RT for 30 min and incubated with an anti-collagen1 primary antibody (cat. no. ab34710; 1:200; Abcam) overnight at 4°C. After washing with PBS, cells were incubated with a FITC-conjugated goat anti-rabbit secondary antibody (cat. no. ab6717; 1:5,000; Abcam) in the dark at 37°C for 1.5 h. After washing with PBS, the slides were incubated with DAPI (RT, 5 min) for nuclear staining. Stained slides were observed using a confocal fluorescent microscope (magnification, ×400).

The levels of the proinflammatory cytokines, TGF-β (cat. no. ab100647), interleukin (IL)-1β (cat. no. ab100562), platelet derived growth factor (PDGF; cat. no. ab184860) and epidermal growth factor (EGF; cat. no. ab217772) were detected using ELISA kits from Abcam according to the manufacturer's protocol. Briefly, standard samples and cell supernatants were added into wells with corresponding antibody coating and incubated for 2 h at room temperature. Subsequently, conjugate was added to the wells and incubated for 2 h at room temperature, followed by 50 µl stop solution to terminate the reaction. Absorbance was measured at a wavelength of 450 nm using a SpectraMax 340 microplate reader (Molecular Devices LLC). A cellular reactive oxygen species (ROS) assay kit (cat. no. ab186027; Abcam) was used to determine ROS levels. Briefly, transfected cells were plated (4×10⁴/100 µl per well) in a 96-well plate overnight. Subsequently, cells were stained with ROS red stock solution for 30 min at room temperature. Absorbance was measured using a microplate reader and ROS levels were determined by the fluorescence increase at Ex/Em=520/605 nm (19).

All experiments were repeated at least three times and statistical analyses were performed using GraphPrism software (version 5.0; GraphPad Prism, Inc.). Data are presented as the mean ± SD. Comparisons among groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential role of lncRNA AK021443 in hepatic fibrosis in vitro, AK021443 was overexpressed in the LX-2 HSC cell line to observe alterations to cellular molecular processes associated with hepatic fibrosis. The results verified the successful overexpression of AK021443 in cells transfected with pcDNA-AK021443 compared with cells transfected with pcDNA (Fig. 1A).

Cell proliferation was assessed using a CCK-8 assay, flow cytometry and western blotting. At 0, 24, 48 and 72 h post-transfection, cell viability was assessed by performing a CCK-8 assay. The results suggested that AK021443 overexpression significantly increased cell proliferation from 24 h in a time-dependent manner compared with the pcDNA group (Fig. 1B). Flow cytometry was conducted to detect the cell cycle distribution, and the results indicated that pcDNA-AK021443 significantly decreased the proportion of G₁ cells, but significantly increased the proportion of S and G₂ phase cells compared with the pcDNA group (Fig. 2A). Moreover, the protein expression levels of cyclin-dependent kinase 2 (CDK2) and cyclin D1 were significantly increased, whereas p21 expression levels were significantly decreased by pcDNA-AK021443 compared with pcDNA (Fig. 2B). Therefore, the results indicated that AK021443 could increase LX-2 cell proliferation.

Subsequently, to investigate whether AK021443 could induce the activation of LX-2 cells and their conversion into myofibroblasts, the expression levels of proteins associated with epithelial-mesenchymal transition (EMT) and ECM were detected. The protein expression level of E-cadherin was significantly decreased, whereas N-cadherin, vimentin and snail protein expression levels were significantly increased in AK021443-overexpression LX-2 cells compared with pcDNA-transfected cells (Fig. 3). AK021443 overexpression also significantly increased the protein expression levels of TIMP1, collagen1 and ET-1, but slightly reduced the expression levels of MMP2 and MMP9 compared with the pcDNA group (Fig. 4). In addition, IF staining was performed to assess collagen1 expression in LX-2 cells transfected with pcDNA-AK021443. The results suggested that pcDNA-AK021443 increased the levels of collagen1 compared with pcDNA, which was consistent with the western blotting results (Fig. 5). The results demonstrated that AK021443 may function to trigger EMT and ECM deposition in HSCs.

Alterations to the inflammatory response in LX-2 cells were investigated. The results indicated that the generation of IL-1β, TGF-β, PDGF, EGF and ROS was increased by AK021443 overexpression compared with the pcDNA group (Fig. 6). The results suggested that AK021443 might display a stimulatory effect on the release of inflammatory cytokines in LX-2 cells.